鸡HNF4α蛋白片段的原核表达与纯化
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摘要
肝细胞核因子4α(Hepatocyte nuclear factor 4α,HNF4α)作为细胞核受体超家族的成员之一,是肝脏内重要的转录因子,对肝细胞分化和成熟起调控作用。研究以大肠杆菌表达系统表达获得鸡的HNF4α重组蛋白为目的,通过PCR技术分别截取鸡HNF4α蛋白gHNF4α-315和gHNF4α-222两个多肽的基因片段,将其克隆入pColdⅢ质粒,并转化入大肠杆菌BL21(DE3),构建重组表达菌,对重组蛋白多肽的表达条件进行优化,同时通过蛋白的变性和复性与Ni-NTA亲和层析纯化重组HNF4α蛋白多肽。结果显示:BL21-pColdⅢ-gHNF4α-222重组菌在IPTG和15℃的低温条件下成功表达分子量为25 ku的重组蛋白gHNF4α-222,而gHNF4α-315未能表达。在15℃下,以终浓度0.1 mmol/L的IPTG诱导18 h是重组蛋白多肽的表达最佳条件,gHNF4α-222主要以包涵体形式表达;从包涵体纯化得到了纯度在95%以上的可溶性gHNF4α-222重组蛋白,为制备鸡HNF4α抗体,进一步研究鸡HNF4α的功能奠定了基础。
Hepatocyte nuclear factor 4 alpha(HNF4α) is a member of the nuclear receptor superfamily and an important transcription factor in liver, which plays regulatory roles in differentiation and maturation of hepatocyte. The aim of this study was to obtain recombinant chicken HNF4α protein using E. coli. expression system. The gene fragments of two truncated polypeptides of chicken HNF4α protein gHNF4α-315 and gHNF4α-222 were amplified by PCR,and cloned into pCold Ⅲ plasmid, and transformed into E. coli BL21(DE3) to generate two recombinant expression bacteria, respectively. The condition of expression was optimized. Meanwhile, purification of recombinant chicken HNF4α polypeptide was performed through protein denaturation, renaturation and Ni-NTA affinity chromatography. The results showed that the BL21-pColdⅢ-gHNF4α 222 recombinant bacteria induced by 0.1 mmol/L IPTG and the temperature of 15 ℃ successfully expressed a 25-ku polypeptide gHNF4α-222, while gHNF4α 315 was failed to express.The optimal expression condition of recombinant polypeptide gHNF4α-222 was at 15℃ by induction of 0.1 mmol/L final concentration IPTG for 18 h.The gHNF4α-222 expressed mainly in the form of inclusion body. Soluble gHNF4α-222 recombinant protein with 95% purity was purified from inclusion body, which laid the foundation for preparation of antibody to chicken HNF4α and further study of the function of chicken HNF4α.
Hepatocyte nuclear factor 4 alpha(HNF4α) is a member of the nuclear receptor superfamily and an important transcription factor in liver, which plays regulatory roles in differentiation and maturation of hepatocyte. The aim of this study was to obtain recombinant chicken HNF4α protein using E. coli. expression system. The gene fragments of two truncated polypeptides of chicken HNF4α protein gHNF4α-315 and gHNF4α-222 were amplified by PCR,and cloned into pCold Ⅲ plasmid, and transformed into E. coli BL21(DE3) to generate two recombinant expression bacteria, respectively. The condition of expression was optimized. Meanwhile, purification of recombinant chicken HNF4α polypeptide was performed through protein denaturation, renaturation and Ni-NTA affinity chromatography. The results showed that the BL21-pColdⅢ-gHNF4α 222 recombinant bacteria induced by 0.1 mmol/L IPTG and the temperature of 15 ℃ successfully expressed a 25-ku polypeptide gHNF4α-222, while gHNF4α 315 was failed to express.The optimal expression condition of recombinant polypeptide gHNF4α-222 was at 15℃ by induction of 0.1 mmol/L final concentration IPTG for 18 h.The gHNF4α-222 expressed mainly in the form of inclusion body. Soluble gHNF4α-222 recombinant protein with 95% purity was purified from inclusion body, which laid the foundation for preparation of antibody to chicken HNF4α and further study of the function of chicken HNF4α.
引文
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[15]曾发姣,舒燕,缪东,等.pCold TM TF载体原核表达FocA可溶性蛋白的研究[J].湖南农业科学,2016(17):12-14.
[16]PALMER I,WINGFIELD P T.Preparation and extraction of insoluble(inclusion-body)proteins from Escherichia coli[J].Current Protocols In Protein Science,2012,16(4):131-143.
[2]张礼勇.基因工程原理及进展[J].科技资讯,2007(18):7.
[3]吴建祥,李桂新.分子生物学实验[M].杭州:浙江大学出版社,2014.
[4]施巧琴,吴松刚.工业微生物育种学[M].福州:福建科学技术出版社,2013.
[5]纪剑飞,包成刚.包涵体重组蛋白的纯化及复性[J].沈阳药科大学学报,1998,15(4):303-307.
[6]郁建锋,张芸,王中亮,等.鸡HNF1α在大肠杆菌中的表达及其纯化[J].农业生物技术学报,2018,26(3):469-475.
[7]SLADEK F M,ZHONG W,JR J E D,et al.Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily[J].Genes&Development,1990,4(12B):2353-2365.
[8]PALANKER L,TENNESSEN J M,LAM G,et al.Drosophila HNF4 regulates lipid mobilization and beta-oxidation[J].Cell Metabolism,2009,9(3):228-239.
[9]LU H.Crosstalk of HNF4alpha with extracellular and intracellular signaling pathways in the regulation of hepatic metabolism of drugs and lipids[J].Acta Pharmaceutica Sinica B,2016,6(5):393-408.
[10]DHE-PAGANON S,DUDA K,IWAMOTO M,et al.Crystal structure of the HNF4 alpha ligand binding domain in complex with endogenous fatty acid ligand[J].The Journal of Biological Chemistry,2002,277(41):37973-37976.
[11]JANA S,DEB J K.Strategies for efficient production of heterologous proteins in Escherichia coli[J].Applied Microbiology and Biotechnology,2005,67(3):289-298.
[12]MAKRIDES S C.Strategies for achieving high-level expression of genes in Escherichia coli[J].Microbiological Reviews,1996,60(3):512-538.
[13]KLERMUND L,RIEDERER A,GROHER A,et al.Highlevel soluble expression of a bacterial N-acyl-d-glucosamine 2-epimerase in recombinant Escherichia coli[J].Protein Expression and Purification,2015,111:36-41.
[14]KUDLA G,MURRAY A W,TOLLERVEY D,et al.Coding-sequence determinants of gene expression in Escherichia coli[J].Science,2009,324(5924):255-258.
[15]曾发姣,舒燕,缪东,等.pCold TM TF载体原核表达FocA可溶性蛋白的研究[J].湖南农业科学,2016(17):12-14.
[16]PALMER I,WINGFIELD P T.Preparation and extraction of insoluble(inclusion-body)proteins from Escherichia coli[J].Current Protocols In Protein Science,2012,16(4):131-143.